Gene cassettes of class I integron-associated with antimicrobial resistance in isolates of Citrobacter spp. with multidrug resistance.

Background and Objectives
Integrons play a major role in the transmission and accumulation of resistance factors in multidrug resistant bacteria. This study was aimed to evaluate the gene cassettes of class I integron and antimicrobial resistance in isolates of Citrobacter with multidrug resistance (MDR).


Materials and Methods
Ninety isolates of Citrobacter spp. were collected from the largest hospital in Kermanshah, Iran. Antimicrobial resistance patterns were determined using disc diffusion method. The class I integron were detected by PCR. The integrase positive isolates were further analyzed for the presence of gene cassettes using 5' and 3' conserved sequences (CSs) primers and PCR products were sequenced. The data were analyzed using the chi-square test.


Results
Of 90 Citrobacter isolates, 46 (51.1%) were multidrug resistant. Class I integron and gene cassettes were determined in 30 isolates (65.2%). Gene cassettes were found which contained genes encoded resistance to aminoglycosides and trimethoprim and a putative gene. Gene cassettes of dfrA12-orfF-aadA2, dfrA1-aadA1, aadA1 and dfrA15-aadA2 were also found in Citrobacter isolates.


Conclusion
Our results indicate there is a high frequency of class I integron among multi-drug resistant strains of Citrobacter isolated from clinical settings. A high frequency of class I integron associated gene cassettes, in particular dfr and aadA, present in MDR strains of Citrobacter. This data indicates an important role of integrons in the creation and transmission of MDR strains in health care centers.


INTRODUCTION
Citrobacter species have been reported as nosocomial pathogens with multidrug resistance (MDR) in many countries since recent decades (1). Citrobacter species have involved in various human infections, in particular, urinary tract infections, wound infec-tions and respiratory infections (2). The antibiotic resistance of this group of bacteria have increased and MDR isolates have frequently been reported (2). The horizontal gene transferring plays the main role in the spread of antibiotic resistance genes and subsequently the rapid emergence of antibiotic resistance among Enterobacteriaceae (3). The mobile genetic elements such as plasmids, transposons and integrons are main factor for horizontal spreading of resistance genes (3). Integrons are conserved DNA sequences, which can efficiently acquire and transfer the resistant genes among bacteria and usually located on mobile genetics elements (4). There are several different classes of integrons, each encodes a distinct integrase gene (4). Class I integron is the most common type presented in clinical isolates of the Enterobacteriaceae (5). It is capable to carry single or multiple gene cassettes, which confer resistance to various antibiotics including, aminoglycoside, β-lactams, chloramphenicol, quinolones and trimethoprim (6). Class I integron has two conserved segments; 5′-CS and 3′-CS, separated by a variable region, included the integrated gene cassettes (7). The 5′-CS encodes integrase, located next to the recombination site (att1) recognized by the integrase and the promoter (P) which controls the transcription of integrated gene cassettes (7). The 3′-CS usually includes truncated qacE (qacED1) and sul1 genes that confer resistance to quaternary ammonium compounds and sulfonamides, respectively (7). Recombination between the attl of integron and attC sites of gene cassettes leads to the insertion of gene cassettes downstream to the resident promoter mediated by integrase (7). Integrase is a member of the tyrosine site-specific recombinase family that catalyze the excision and integration of DNA fragments, including gene cassettes (7). Near two hundreds of different cassette arrays have been identified that are flanked by the 5΄-CS and 3΄-CS endes (6). A strong association of integrons associated gene cassettes with MDR isolates of Enterobacteriaceae has been found (8,9). Gene cassettes encode resistance to various antimicrobial agents, including dihydrofolate reductases (dfr), chloramphenicol acetyl-transferases (cat, cml), β-lactamases (bla), aminoglycoside-modifying enzymes (aac, aad, aphA) and ADP-ribosyl transferases (arr) have been frequently identified within integrons (8,9). This study aimed to evaluate the gene cassettes of class I integron-associated antimicrobial resistance in isolates of Citrobacter with multidrug resistance (MDR).

MATERIALS AND METHODS
Bacterial isolates. In this descriptive study, 288 different clinical samples (e.g., wound, blood, urine, stool, and other samples) from patients admitted in the largest hospital in Kermanshah were collected during 2014-2015. Using the bacteriological and API20E Kit (bio-Merieux, France) testing, 90 Citrobacter isolates were confirmed.

Polymerase chain reaction-detection of class I integron.
The presence of class I integron was screened by PCR using intIF and intIR primers (SinaColon, Iran) ( Table 1). Each single reaction mixture (25 μl) contained 2μl of DNA suspension, 10 pmol of each primer, 2x GoTaq Green Master Mixture (SinaColon, Iran). The PCR conditions were as follows; 94°C for 5 minutes, followed by 35 cycles at 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 45 minutes and final extension at 72°C for 5 minutes.
Detection of the variable region of class I integrons. PCR was performed with class I integrase positive isolates using two primers 3'CS and 5'CS (SinaColon, Iran) ( Table 1) to amplify the variable region of integron. Each single reaction mixture (25μl) contained 2 μl of DNA suspension, 10 pmol of each primer, 2 x GoTaq Green Master mixtures. PCR reactions began with 5 min of primary denaturation at 94˚C followed by 35 cycles of 94˚C for 1 min, 58˚C for 1 min and finally 72˚C for 1 min. The final extension was performed at 72˚C for 10 min. After electrophoresis of PCR products on 1% agarose gel (Merck Co, Germany) and staining with ethidium bromide, the gels were visualized by Gel-Documentation apparatus (Bio Rad, USA).

DNA sequence analysis.
A number of PCR products with sharp bands were cut and purified using the QIA quick PCR purification Kit (QIA-GEN, Germany) followed by sequencing. The DNA sequences were performed using an ABI 3730XL DNA analyzer (Macrogen Inc., Korea). Sequences were analyzed using BLAST search (http://www.ncbi.nlm.nih.gov/BLAST).

Statistical analysis.
Data were recorded and entered into an Excel file. Statistical analyses were performed using SPSS software (Version 20). Variables were analyzed by the Chi-square test. A p-value of < 0.05 was set as the statistical significance of all analyses.

DISCUSSION
Recent research shows an increase of Citrobacter isolates among urinary tract infection agents with high antibiotic resistance in developed countries (12)(13)(14). Our results of antibiotic susceptibility testing showed the highest resistance of Citrobacter isolates to cefazolin and ampicillin and highest sensitivity to carbapenems, tazobactam and gentamicin. These findings are consistent with the results of previous studies (15)(16)(17). A high percentage of Citrobacter isolates in Kermanshah with multi-drug resistance indicates the dissemination of antibiotic resistance genes in this opportunistic pathogen (7). On the other hand, the accumulation of resistance genes within integrons con-

Integron-positive isolates
Integron-negative isolates tributes to the spread of MDR strains among Enterobacteriaceae isolates (18). Class I integron is widely distributed among multidrug resistance of Enterobacteriaceae isolates (19). Studies in Malaysia and Egypt have reported the rate of class I integron in isolates of Citrobacter and Enterobacteriaceae with 50% and 51%, respectively (5,20). The above results are compatible with our results for the frequency of class I integron among Citrobacter isolates in Kermanshah. Class I integron has been found to carry resistance to several antimicrobial agents in bacteria. For instance, cassettes for resistance to fluoroquinolones, β-lactams, aminoglycosides, trimethoprim and chloramphenicol have been identified (5). According to research data, most of the resistant genes for aminoglycosides (aad, aac) are transmitted by class I integrin (21). The results of our study also suggest a statistically significant association between the presence of class I integron and resistance to streptomycin. In our study, a significant correlation between the presence of class I integron and resistance to ciprofloxacin was also noted, which is consistent with other studies (21,22). Although the mutation in topoisomerase genes is the main mechanism of resistance to fluoroquinolones, recently proteins have been identified encoded by integrons and carried on plasmids which increases the bacterial permeability for quinolones (23,24).
Gene cassettes with different sizes carried by class I integron in Citrobacter isolates are consistent with the results of other studies on Enterobacteriaceae family (25,26). Similar to other studies, our results indicate that integrons can carry several cassettes simultaneously and contribute to the emergence of MDR strains (27,28). In our study, the phenotypic resistance to the certain antibiotics was observed in isolates carried the corresponding gene cassettes. For instance, there is a significant association between the presence of dehydrofolate reductase and aminoglycoside adenyltransferase cassettes with phenotypic resistance to trimethoprim-sulfamethoxazole and streptomycin, respectively. This indicates the expression of integron genes and their role in the phenotype of bacteria.
Our results show eight different patterns of class I integron gene cassettes. The DNA analysis of gene cassettes indicates several antibiotic resistance gene cassettes. Two variants of aminoglycoside adenyl transfers (aadA1/aadA2) were detected which encode aminoglycoside 3'-9-adenylyltransferases and confer the resistance to streptomycin and spectinomycin (29). Sequence analysis also revealed three variants of dfrA (dfrA1/dfrA12/dfrA15), which encode the dihydrofolate reductase gene, confer resistance to trimethoprim (29). The horizontal transmission of resistance genes between bacteria can occur and expand the gene cassettes (30). As indicated by our results and also supported by other studies, the dfr cassette is mostly as-sociated with the aadA gene cassette (30). These observations suggest this combination of gene cassettes can reflect their co-transmission and stable integration (31). In the present study, the dfrA1-aadA1 gene cassettes showed a high prevalence in Class I integron, which is consistent with the results of other studies on Enterobacteriaceae isolates (32,33). According to the previous research, Class I integron contained the aadA1 or dfrA1-aadA1 cassettes are commonly found in E. coli isolates in Europe (34)(35)(36). Similarly, these gene cassettes have been reported in Asian countries (37,38). In some studies in Iran, the 5-arr, aacA4-orfD, aadA5-dfrA17, dfrA1, aadA1-dfrA1 and aadA2-dfrA12-orfF cassettes have been reported as the most common cassettes in Klebsiella pneumoniae and E. coli isolates (29,(39)(40). It seems that the aadA and dfrA gene cassettes in Iran are also prevalent In conclusion, our results indicate a high prevalence of MDR among Citrobacter isolates in Kermanshah. A high frequency of class I integron and the associated gene cassettes, in particular dfr and aadA, present in MDR strains of Citrobacter isolated from hospitalized patients. They may play an important role in the creation and transmission of MDR strains. Statistical analysis indicates the association of integration class I and MDR isolates in this opportunistic pathogen, which needs continues surveillance in health care centers.